Codon-Optimized Oligonucleotide for Induction of Elastin De-Novo Synthesis in Mammals

This application is a continuation of copending international patent application PCT/EP2023/081133 filed on 8 Nov. 2023 and designating the U.S., which has been published in English, and claims priority from European patent application EP 22 208 908.8 filed on 22 Nov. 2022. The entire contents of these prior applications are incorporated herein by reference.

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “5402P658USCONWO_sequence listing”, created Nov. 18, 2022, which is 32 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

The present invention relates to oligonucleotides for induction of elastin de-novo synthesis in mammals, a method of treatment of diseases and medical conditions associated with insufficient tissue elasticity, use of the oligonucleotide in such treatment, as well as pharmaceutical and cosmetical compositions comprising such oligonucleotide.

The present invention relates to the field of molecular medicine, especially to the protein expression for therapeutic or cosmetic purposes, more specifically to the induction of de-novo synthesis of desired extracellular matrix proteins in mammals.

Elastin is a component of the extracellular matrix (ECM) of vertebrates and provides elasticity and flexibility to tissues. Elastin fibers are built by covalently crosslinking lysine residues of the elastin precursor tropoelastin (TE). During ontogenetic development, the soluble monomer TE is secreted by cells, such as smooth muscle cells, fibroblasts, and endothelial cells, and assembled into highly stable, insoluble, polymeric elastin fibers in the ECM by the enzyme lysyl oxidase. The process of generating the elastin fibers will be referred to herein as elastogenesis.

TE expression is largely restricted to the fetal phase and the early postnatal years. From adolescence onward, elastin synthesis decreases and ceases in adults. Although the half-life of elastin is approximately 74 years, loss of elastin fibers is caused by age-related degradation, disease, or injury and can subsequently lead to loss of tissue elasticity, flexibility and thus, integrity and functionality.

Elasticity and thus elastin is of high importance in multiple organs as for example, the lung, the heart, the skin, or blood vessels, especially also the aorta. Various inherited diseases, such as Williams-Beuren syndrome (WBS) or cutis laxa, result in impaired elastogenesis and lead to loose skin and vascular defects, such as supravalvular aortic stenosis. In the skin, damage to elastin fibers due to injuries, diseases, sunburn, and age-related degradation results in irreversible loss of skin elasticity. The loss of elastin in the dermis after severe burns results in significant physical damage such as scarring, wound contraction, and loss of skin extensibility. Thus, the regeneration of elastin fibers, plays a crucial role in wound healing and scar formation, as well as in restoring the functionality and elasticity of the skin.

In the above-mentioned cases, it is of interest to be able to induce denovo synthesis of elastin. Various strategies have been applied to restore skin elasticity by inducing elastogenesis. The most prominent ones are the use of viral vectors (Xiong, J., et al., Elastic fibers reconstructed using adenovirus-mediated expression of tropoelastin and tested in the elastase model of abdominal aortic aneurysm in rats. J Vasc Surg, 2008. 48(4): p. 965-73) and the use of synthetic mRNA (DE102013005361A1, Lescan, M., et al., De Novo Synthesis of Elastin by Exogenous Delivery of Synthetic Modified mRNA into Skin and Elastin-Deficient Cells. Mol Ther Nucleic Acids, 2018. 11: p. 475-484).

The therapeutical application of synthetic mRNA has proven most promising and gained high interest due to several advantages: First, it can be produced easily by in vitro transcription (IVT). Second, it is not integrated into the host genome, and it underlies a physiological decay. Therefore, it persists only transiently in the cell and greatly reduces the mutagenic risk as compared to viral vectors. Also, other side effects, associated with long-term protein overexpression are avoided. Third, it can be more easily delivered into cells due to the smaller size compared to plasmids or viral vectors.

DE102013005361A1 discloses a synthetic mRNA encoding elastic fiber proteins, one of which is elastin, and comprising nucleotide analogues. However, the enhancement of elastin expression is limited and requires the application of a high amount of at least 5 μg of mRNA.

Lescan et al., 2018 disclose an increased elastin synthesis in various human cell types after the transfection with synthetic TE mRNA and in ex vivo porcine skin after intradermal microinjection of the same.

Challenges in the therapeutic application of RNA are its biological instability and immunogenicity, limiting its bioavailability and applicability, respectively. Enzymes degrading RNA are almost ubiquitously present, especially in the extracellular space. Native immune mechanisms involving Toll-like receptors (TLR) recognize singlestranded RNA (TLR-7, TLR-8) or double-stranded RNA (TLR-3), which subsequently induces an inflammatory immune response.

Thus, it is an object of the present invention to provide an oligonucleotide with which the disadvantages of the prior art are eliminated or at least mitigated. Especially such an oligonucleotide should be provided which is able to induce de-novo synthesis of elastin in mammalian cells and tissues by enhancing the expression efficiency of a synthetic mRNA encoding TE. This object is fully solved by the present invention.

In an aspect of the invention, the above-identified disadvantages are overcome by providing an oligonucleotide comprising a nucleotide sequence encoding a TE protein, characterized in that the nucleotide sequence is codon-optimized for expression in a mammal cell.

Herein, as generally in the field, codon-optimization is to be understood as the exchange of codons to which at least one synonymous codon exists by a synonymous codon that is expected to yield a higher expression efficiency. The expectation of which codon will render the expression efficiency higher is dependent on various factors, the most commons of which are the species and the GC content of the codon and the total sequence. The codon-optimization can be parametrized as the Codon adaptation index (CAI) (Sharp, P. M. and W. H. Li, The codon Adaptation Index—a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res, 1987. 15(3): p. 1281-95). The CAI represents the geometric mean of all amino acids regarding the fraction of codons within all synonymous codons encoding a certain amino acid, that are identical to the one of all synonymous codons encoding a certain amino acid, that is most often used for encoding said amino acid within a reference set of genes.

As this has been realized by the inventors, codon-optimization can positively affect the expression efficiency and stability of synthetic mRNA enoding TE, leading to increased protein expression levels (Presnyak, V., et al., Codon optimality is a major determinant of mRNA stability. Cell, 2015. 160(6): p. 1111-24). This finding was especially surprising for the TE and not to be expected for the following reasons.

One of the most important measures during the design of synthetic mRNA is typically the GC-content. It should not be too low, decreasing expression efficiency due to enhanced degradation, and not too high, enhancing the probability of secondary structures within the RNA molecule limiting its accessibility for the translation machinery.

Codon-optimized synthetic mRNAs have been disclosed in studies concerning the expression of proteins that do not have a high GC-content in their encoding wildtype nucleotide sequence. U.S. Pat. No. 10,898,584B2 discloses synthetic mRNAs relying on strong enhancement of the GC content. Codon-optimized synthetic mRNAs are disclosed with respect to non-fiber building, non-tissue-structure and non-ECM proteins such as interferon (IFN)-α and erythropoietin (EPO) (Kariko, K., et al., Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin. Mol Ther, 2012. 20(5): p. 948-53, and Hochmann, S., et al., Evaluation of modified Interferon alpha mRNA constructs for the treatment of non-melanoma skin cancer. Sci Rep, 2018. 8(1): p. 12954).

The prior art teaches the necessity to enhance the GC content for enhancing the expression efficiency of a synthetic mRNA. Hitherto, it has been considered to be not possible to enhance the expression efficiency of proteins as TE, which has already in its human wildtype sequence a high GC-content of 64.3%. Differently speaking 76% of all amino acids of the human TE protein comprise glycine (29%), alanine (22%) valine (13%), and proline (12%). All of these 4 amino acids have GC rich codons and therefore, at least those 76% of the respective codons are not optimizable with respect to their GC content. Of the remaining 24% some do by nature not have synonymous GC rich codons or if available, those may be inconvenient for expression in humans or other mammals.

Surprisingly, the inventors were able to codon-optimize a wildtype human nucleotide sequence encoding TE, which naturally has a high GC-content, such that the expression efficiency is enhanced, without adhering to the dogma of enhancing the GC-content. Advantageously, this adds an additional degree of freedom to the generation of such optimized sequences.

In an embodiment of the present invention, the oligonucleotide is an oligoribonucleotide, preferably an mRNA.

By choosing the oligonucleotide to be a ribonucleic acid, a permanent introduction of the oligonucleotide into the genome of the respective cells is avoided, greatly reducing a mutagenic risk. Furthermore, as elastin has an extraordinarily long halflife, the transient presence of the mRNA within the cells is advantageous as it allows the cells to return to their physiological status of not producing elastin. Thus, highly specifically, the desired result of depositing additional elastin fibers in the ECM can be achieved without permanently altering the behavior of the respective cells.

Herein, the terms mRNA and synthetic mRNA are used interchangeably. The term “synthetic” shall make clear that the respective mRNA is artificially produced, preferably by in-vitro transcription (IVT). There may be or may be no structural or chemical differences between a synthetic mRNA and a mRNA.

In another aspect of this embodiment, the oligonucleotide, comprises a 5′-Cap-structure, which is preferably selected from the group consisting of: 3′-O-Mem7G(5′)ppp(5′)G, m7G(5′)ppp(5′)(2′OMeA)pG, m7G(5′)ppp(5′)(2′OMeA)pU, m7(3′OMeG) (5′)ppp(5′)(2′OMeA)pG, and/or a polyA-tail, preferably consisting of at least approx. 70 adenine nucleotides, further preferably of approx. 120 adenine nucleotides. The 5′-Cap-structure can be of natural or synthetic or modified origin. Therefore, the term “5′-Cap-structure” refers to any natural 5′-Cap-structure naturally used by eucaryotic cells as well as to any synthetic/modified not naturally occurring 5′-Cap-structure suitable for replacement of natural 5′-Cap-structures in view of function and cytotoxicity.

Such modifications mimic the natural structure of mammalian mRNA and thus advantageously provide a certain degree of stability to the oligonucleotide such that the oligonucleotide is degraded slower by the respective cell it is delivered to, allowing the oligonucleotide to induce TE synthesis.

While the nucleotides can be naturally occurring nucleotides, i.e., nonmodified, in a further embodiment of the invention, at least one of the nucleotides is an analogue of a naturally occurring nucleotide. Advantageously, this further decelerates the degradation of the synthetic mRNA and prohibits the synthetic mRNA to be recognized by the immune system, such that an inflammatory response is avoided. In this embodiment, a substantial fraction of all nucleotides is an analogue of the respective natural nucleotide. Natural nucleotides are meant to be nucleotides comprising nucleobases naturally occurring in mammalian DNA or RNA. Specifically, those include adenine, guanine, cytosine, thymidine, and uracil. An analogue is to be understood as comprising a chemical structure similar to a natural nucleotide, allowing the cellular translation machinery to translate the nucleotide sequence encoded by the oligonucleotide into an encoded protein while decelerating degradation and/or reducing immunogenicity of the respective oligonucleotide.

The term “substantial fraction” means that not necessarily all of a sort of natural nucleotide need to be replaced by an analogue. At least 5% or more of one sort of natural nucleotide are replaced by an analogue, preferably 25% or more, more preferably 100%.

In this embodiment, an analogue can be for example pseudouridine, N1-Methylpseudouridine, 5-Methylcytidine, phosphorothioate, phosphoramidate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 2-thiouridine, 5-methyluridine, 5-methyluridine-5′-triphosphate (m5U), 5-iodouridine-5′-triphosphate (15U), 4-thiouridine-5′-triphosphate (S4U), 5-bromouridine-5′-triphosphate (Br5U), 2′-methyl-2′-deoxyuridine-5′-triphosphate (U2′m), 2′-amino-2′-deoxyuridine-5′-triphosphate (U2′NH2), 2′-Azido-2′-deoxyuridine-5′-Triphosphate (U2′N3), 2′-Fluoro-2′-deoxyuridine-5′-triphosphate (U2′F), inosine, 3-methylcytidine, 2-thiocytidine, 2′-methyl-2′-deoxycytidine-5′-triphosphat (C2′m), 2′-amino-2′-deoxycytidin-5′-triphosphate (C2′NH2), 2′-fluoro-2′-deoxycytidine-5′-triphosphate (C2′F), 5-iodocytidin-5′-triphosphate (15U), 5-bromocytidine-5′-triphosphate (Br5C), and 2′-azido-2′-deoxycytidine-5′-triphosphate (C2′N3), and is preferably selected from the group consisting of: pseudouridine, N1-methylpseudouridine and 5-methylcytidine. Those analogues have turned out to be highly suitable for reduction of immunogenicity of the respective oligonucleotide while enhancing the expression efficiency.

In another embodiment of the present invention, the TE protein comprises the amino acid sequence of SEQ ID NO: 1.

Advantageously, the use of this human TE sequence allows to express TE in a human cell such that the oligonucleotide can be used in medical or cosmetical application targeting humans.

In another embodiment of the present invention, the oligonucleotide comprises a nucleotide sequence of any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

Those sequences are codon-optimized variants of the wildtype sequence. Advantageously, those sequences show a low immunogenicity and a high expression efficiency.

Hereinafter, the term “expression efficiency” is to be understood as the quotient from the amount of elastin protein synthesized by the respective mammalian cells or tissue within a certain period to the amount of oligonucleotide applied. In this context the term “applied” is to be understood as any type of method employable, resulting in or comprising the introduction of DNA or RNA into cells.

In another embodiment of the present invention, the oligonucleotide can be used in the treatment of diseases and medical conditions associated with insufficient tissue elasticity. Such use advantageously allows to induce a transient de-novo synthesis of elastin fibers within the ECM of a tissue of a mammal and thus to enhance the elasticity of the tissue. Preferably such mammal is a human, a mammal being property of a human or a domesticable mammal, more preferably such mammal is a human.

Herein, the term “disease” is to be understood as generally in medicine as any kind of unphysiological condition of the respective mammal, which may be caused for example genetically, oncologically, microbially, bacterially, virally, or psychologically. The term “medical condition” is to be understood as referring to any kind of unphysiological condition, which may be caused for example by physical damage of the mammal's body, or aging. However, as generally understood “medical condition” may also refer to diseases resulting from such damage and “disease” may refer to a medical condition. Therefore, the terms “disease” and “medical condition” can be used synonymously herein.

A disease or medical condition associated with “insufficient elasticity” is to be understood as any disease or medical condition affecting a mammal, regarding which the mammal may benefit from an enhancement of the elasticity of any of its tissues. Elasticity in this context means the ability of a tissue to resist a distorting influence and to return to its former size and shape when that influence is removed.

In this embodiment, the treatment is preferably selected from the group consisting of: therapies of genetic defects of elastin-synthesis, arteriosclerosis, aortic stenosis, aortic and/or aneurysm, chronic obstructive pulmonary disease (COPD), ocular diseases, e.g. AMD, aortic valve insufficiency, dermatochalasis, Williams-Beuren Syndrome, cutis laxa, ligament disorders, subvalvular congenital aortic stenosis (SVAS), scarred tissue, and for scar-free wound-healing. Mammals suffering from those diseases or medical conditions can benefit from enhanced tissue elasticity.

In another aspect, the present invention relates to a pharmaceutical composition comprising the oligonucleotide according to the invention and a pharmaceutically acceptable carrier.

A suitable carrier for the specific treatment, will be clear to the skilled person. Additional disclosure of pharmaceutically acceptable carriers of synthetic mRNA can be found for example in Ouranidis A, et al., mRNA Therapeutic Modalities Design, Formulation and Manufacturing under Pharma 4.0 Principles. Biomedicines, 2021. December 27; 10(1): 50.

In one embodiment, the pharmaceutical composition is configured for a systemic administration onto the mammal, wherein preferably the systemic administration is a parenteral route of administration, more preferably an intravenous injection. The systemic administration can be any enteral or parenteral route of administration, preferably a parenteral route of administration, more preferably an intravenous injection. Advantageously, a systemic administration allows to treat the whole organism, which may be especially advantageous for treatment of diseases or medical conditions associated with insufficient tissue elasticity of blood vessels.

In one embodiment, the pharmaceutical composition is configured for an administration locally by injection into or external application onto the tissue of the mammal, wherein preferably the pharmaceutical composition is present in a formulation or delivery form selected from the group consisting of: a creme, a gel, a liquid, a paste, a spray, a plaster, a microneedle, a medical bandage, a facemask, an implant, and a stent.

A local administration is advantageous since it allows to specifically induce de-novo synthesis of elastin at the site of disease or medical condition without unnecessarily affecting other regions of the body not requiring additional elastin fiber deposition, which may cause harm to the respective mammal.

Any type of external application further is advantageous in that it is less invasive and thus less stressful than other modes of application.

The above-mentioned formulations or delivery forms of a creme, a gel, a liquid, a paste, a spray, and a plaster are advantageous because they allow the application of the pharmaceutical composition on any region of the body, no matter the size of the region.

Advantageously, the formulation or delivery form of a plaster or microneedles allows to apply the pharmaceutical composition to a very concise and defined region of the body in need of it, wherein the respective region can be exposed to the pharmaceutical composition for a longer period and on body regions usually covered by clothes, which otherwise may hinder the application of the pharmaceutical composition in the quotidian.

The formulation or delivery form of a bandage or facemask is advantageous in that it allows an even and prolonged distribution of the pharmaceutical composition on the face or another larger, for example burned, region of the body.

The formulation or delivery form of an implant or a stent advantageously allows to apply the pharmaceutical composition to blood vessels and/or local tissue areas.

In another aspect the present invention relates to a cosmetical composition comprising the oligonucleotide according to the invention for use in the enhancement of human tissue elasticity, preferably in the treatment or prophylaxis of wrinkles formed on human skin. The cosmetical composition can induce de-novo-synthesis of elastin fibers within the skin of humans, which can restore or maintain sufficient elasticity of the skin to avoid wrinkles and the like.

In another aspect the present invention relates to a cosmetical method for induction of a de-novo synthesis of elastin in the tissue of a mammal comprising application of the cosmetical composition locally by injection into or external application onto the tissue.

In one embodiment of the present invention, when performing the cosmetical method, the application is repeated at least once. A repeated application of the cosmetical composition comprising the oligonucleotide allows to accumulate elastin fibers within the respective tissue, which may be necessary or desired since an oligonucleotide will transiently and non-permanently cause de-novo synthesis of elastin fibers.